JP2002541779A - Cloning method of signaling intermediate - Google Patents

Abstract

(57) Summary The present invention is a method for identifying a polypeptide that increases gene expression from a promoter, comprising the following steps: (a) the library comprises gene expression from the promoter; Expressing a recombinant anti-cell death gene such that the expression of the reporter gene is increased if it includes a polypeptide that increases the expression of the reporter gene, and operably linked to a promoter (as shown in the figure). Contacting the cell with the polypeptide library; (b) determining whether expression of the reporter gene in the cell has been increased as a result of contacting the polypeptide library; and (c) increasing reporter gene expression If so, identifying polypeptides in the library that increase reporter gene expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

Statement as a Federally Sponsered Research This work was completed with the support of the National Institutes of Health under the approval number AI27849. The United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION The present invention relates to methods for identifying polypeptides and compounds that regulate gene expression.

[0003] Pharmaceuticals have historically been derived from libraries of thousands of compounds, usually one at a time.
One compound has been developed by testing in laboratory animals. The slow progression of this method, and its incompatibility with the screening of large numbers of different compounds, has led to the development of an assay-based approach that can be done quickly and ex vivo. These approaches have evolved drug development methods into a chain of several phases, sometimes partially parallel.

[0004] In the first phase, target identification and validation, potential drug candidate targets are identified by various means. These tools include studies of the pathophysiology of disease in humans or experimental animals, analysis of candidate signaling pathways in vitro, natural experiments such as genetic diseases in humans or other animals, targeted or random in model organisms. Hypotheses formed as a result of severe gene disruption or from disclosure by a competitor. Thereafter, the expected effects of hypothetical agents that interact with the target of interest to stimulate, block, or alter the target activity are tested. A target is said to be validated if the tests indicate that the drug acting on the target is considered to have the desired physiological significance to the intact organism. Formally, no identification or demonstration of the target was required, and in fact, neither of these phases existed in historical methods.

[0005] Current models of pharmaceutical industrial production emphasize the importance of efficiently identifying a number of demonstrated targets and developing rapid screening methods for their activity. Since the targets cannot be predicted efficiently, the popular belief that a large number of targets must be evaluated in order to develop a small number of drugs is driving this model. Thus, there remains a significant need in the industry to accelerate the speed of target discovery and demonstration.

[0006] Once the target has been identified and validated, assays must be developed to test the ability of large libraries of synthetic or natural compounds to interact with the target. When a compound that specifically acts on a target is identified, it is usually
Called hit. The definition of a hit generally includes compounds that pass various secondary tests that confirm that their activity is specific for the target of interest. From the various hits generated, one or more structures are selected that serve as a starting point for a systematic modification program of the chemical structure. These structures are called lead compounds, which are often selected from hits based on compatibility with direct synthesis programs, expected toxicity, or expected absorption, distribution, metabolic or excretion properties. Furthermore, while this step involves a wide range of practices and is usually distinguished, hit and lead compounds are sometimes referred to as if they were equivalent.

SUMMARY OF THE INVENTION In general, the present invention provides a novel and rapid method for identifying selected signaling pathway members that are targeted for drug design.

[0008] In a first aspect, the invention features a method of identifying a polypeptide that increases gene expression from a promoter, comprising: (a) a polypeptide that increases gene expression from a promoter Contacting a library of polypeptides, wherein expression of a reporter gene is increased when is included in the library, with cells containing a reporter gene expressing a recombinant anti-cell death gene and operably linked to a promoter. (B) determining whether the expression of the reporter gene in the cell has been amplified as a result of contacting the polypeptide library; and (c) amplifying the reporter gene expression if the expression of the reporter gene is increased; Identifying the polypeptides of the library.

In a second aspect, the invention features a method of identifying a polypeptide that reduces gene expression from a promoter, comprising the following steps: (a) a polypeptide that reduces gene expression from a promoter Contacting a polypeptide library expressing a recombinant anti-cell death gene and comprising a reporter gene operably linked to a promoter, wherein expression of the reporter gene is reduced when is included in the library. (B) determining whether the expression of the reporter gene in the cell has decreased as a result of contacting the polypeptide library; and (c) determining whether the expression of the reporter gene in the library has decreased if the expression of the reporter gene has decreased. Identifying the polypeptide to be reduced.

In a third aspect, the invention features a method for identifying a polypeptide that alters activation of a transcription factor activation domain, comprising the steps of: (a) expressing a reporter gene by a promoter Is altered when a polypeptide that alters activation of the transcription factor activation domain is included in the library, fused to a recombinant anti-cell death gene and a mammalian transcription factor activation domain. A cell containing a reporter gene operably linked to a basal promoter and a promoter comprising a binding site for the DNA-binding domain, which expresses a chimeric transcription factor comprising a yeast or bacterial DNA-binding domain. Contacting a library of polypeptides; (b) contacting with the polypeptide library results in a cell Phase expression of transporter gene to determine whether or not they have changed; and (c) when the expression of the reporter gene is modified, modifying the reporter gene expression, identifying a polypeptide from the library.

[0011] In a fourth aspect, the invention features a method of identifying a compound that alters gene expression from a promoter, comprising the following steps: (a) wherein the compound that alters gene expression from a promoter is live Contacting the compound library with a cell expressing a recombinant anti-cell death gene and comprising a reporter gene operably linked to a promoter, wherein the expression of the reporter gene is altered when included in the rally; (b ) Determining whether the expression of the reporter gene in the cell has been altered as a result of contacting the compound library; and (c) if the expression of the reporter gene has been altered, modifying the reporter gene expression from the library Identifying the compound of

In a fifth aspect, the invention features a method of identifying a compound that reduces gene expression, comprising the steps of: (a) (i) a recombinant anti-cell death gene; A second gene encoding a peptide;
And (iii) contacting a compound library with a cell that expresses a reporter gene that reduces expression when the function of the polypeptide is inhibited; (b) contacting the compound library with the cell, resulting in expression of the reporter gene Determining whether or not is reduced; and (c) identifying a compound from the library that reduces reporter gene expression if the reporter gene expression is reduced.

In a preferred embodiment of the first, second and third aspects, the library of DNA molecules encoding the polypeptide library is expressed in a cell. The cell can be the same cell that expresses the recombinant anti-cell death gene and contains a reporter gene operably linked to a promoter (in this case, expressing the recombinant anti-cell death gene, The polypeptide is produced by a cell similar to a cell containing a reporter gene operably linked to a promoter), or contains a reporter gene that expresses a recombinant anti-cell death gene and is operably linked to a promoter The polypeptide can be produced by a cell other than the cell. Preferably, the DNA molecule is expressed from a high efficiency expression system.

In another preferred embodiment of the first, second and third aspects, the library of DNA molecules is introduced into cells by transfection, and the average number of DNA molecules introduced into cells by transfection is , At least 25. Preferably, the average number of DNA molecules introduced into the cells by transfection is at least 100, even 150.

[0015] In still another preferred embodiment of the first, second and third aspects, the polypeptide is selected from the group consisting of an extracellular ligand, a cell surface receptor, and a signaling intermediate, and the DNA molecule is Is expressed from a high efficiency expression system.

In a preferred embodiment of the first, second, third, fourth and fifth aspects, step (c) comprises the following steps: (i) two or more such less complex libraries (Ii) repeating steps (a) and (b) until a polypeptide that activates reporter gene expression is identified. Also, the promoter can be from a mammal, and the polypeptide library can include bacterial or viral polypeptides.

In another preferred embodiment of the first, second, third, fourth and fifth aspects, the promoter is a heterologous promoter, the reporter gene is GFP, and the anti-cell death gene is a bc1 family member, The cell is selected from the group consisting of IAP family members and crmA, and the cell is selected from the group consisting of CHO, CD-1, Cos, 293, HeLa, BHK or L cells.

In another aspect, the present invention provides a method for determining whether a compound modulates NF-kB biological activity, comprising: (a) providing a cell expressing a BCMA polypeptide; (b) contacting the candidate compound with the cell, and (c) measuring the BCMA polypeptide expression level in the cell, wherein the cell is compared with a cell that has not been contacted with the candidate compound. Changes in BCMA polypeptide expression level indicate candidate compounds as NF-k
Identifying the compound as a modulator of B activity.

In yet another aspect, the invention provides a method for determining whether a compound modulates BCMA biological activity, comprising: (a) providing a BCMA polypeptide;
Contacting the polypeptide with the candidate compound, and (c) measuring the biological activity level of the BCMA polypeptide, wherein the biological activity level of the BCMA polypeptide is compared to a polypeptide that has not been contacted with the candidate compound. Identifying the candidate compound as a compound that modulates BCMA activity. BCMA polypeptides may be intracellular or in a cell-free system. A preferred BCMA biological activity is NF-kB biological activity (eg, regulation of transcription by NF-kB) or regulation of NF-kB expression. Preferably, the BCMA polypeptide comprises a polypeptide sequence having substantial identity to amino acids 98-164 of human BCMA (SEQ ID NO: 1) or amino acids 97-163 of mouse BCMA (SEQ ID NO: 2).

In yet another aspect, the invention provides a method for determining whether a compound modulates NF-kB activity, comprising: (a) providing a BCMA polypeptide; Contacting the peptide with a candidate compound, and (c) detecting the binding between the candidate compound and the polypeptide, wherein the candidate compound that binds to the polypeptide is NF-kB
Being a compound that modulates biological activity. BCMA polypeptides may be intracellular or in a cell-free system. Preferably, the BCMA polypeptide is 98-164 amino acids of human BCMA (SEQ ID NO: 1) or 97 amino acids of mouse BCMA.
163 amino acids (SEQ ID NO: 2).

In another aspect, the invention features a substantially pure polypeptide consisting of a BCMA polypeptide molecule that lacks the BCMA extracellular domain. Preferably, the BCMA polypeptide is 98-164 amino acids of human BCMA (SEQ ID NO: 1) or mouse BCMA
A polypeptide sequence having substantial identity to amino acids 97-163 of SEQ ID NO: 2 (SEQ ID NO: 2). Another preferred polypeptide is a BCMA polypeptide that modulates NF-kB activity.

In yet another aspect, the invention features an NF-kB modulator comprising a BCMA polypeptide covalently linked to a heterologous compound. Preferably, the BCMA polypeptide comprises a polypeptide sequence having substantial identity to amino acids 98-164 of human BCMA (SEQ ID NO: 1) or amino acids 97-163 of mouse BCMA (SEQ ID NO: 2). Preferably, the modulator modulates NF-kB biological activity or expression.

[0023] The present invention also provides a method of contacting a cell with a recombinant BCMA polypeptide having an NF-kB activating effect, or a method of encoding a recombinant BCMA polypeptide having an NF-kB activating effect in a cell. It features a method for activating the NF-kB action of a cell by contacting the nucleic acid molecule.

[0024] In a related aspect, the invention features the use of a BCMA polypeptide and nucleic acid for producing a pharmaceutical composition for treating cancer or apoptosis.

“Reporter gene” refers to a DNA or RNA sequence that encodes a reporter protein that can be readily detected inside or outside a cell.

[0026] The reporter gene is operably linked to a promoter with low spontaneous activation so that the activity of the reporter protein in the presence of the activating polypeptide is
At least 2 standard deviations greater than the activity in the absence of the activating polypeptide. Deletion of a suppressor sequence from a native gene that may be found inside and outside the transcribed portion and that may affect mRNA formation, stability, or translation efficiency; increases the rate of mature mRNA formation The addition of introns that are used efficiently to enhance the activation region or binding site upstream of the well-known transcriptional activator; increase the copy number of the reporter gene; and between the substrate binding region or the chromatin insulator sequence. Various methods are known in the art for increasing the sensitivity of a reporter gene, including protecting the activity of the reporter gene from accidental stimulating or suppressing effects of adjacent DNA by inserting the reporter gene into the DNA.

[0027] Many different types of reporter proteins are well known in the art.
They often contain proteins not normally found in certain cells or present in small amounts. They include enzymes that detoxify antimicrobial agents, such as aminoglycosides or aminocyclitol phosphotransferases or acetyltransferases, β-lactamases or chloramphenicol acetyltransferases; chromogenic reactions, fluorogenic reactions or Enzymes of various origins, such as β-galactosidase, β-glucuronidase, alkaline phosphatase, catechol 2,3-dioxygenase or various peroxidases, which catalyze a chemiluminescent reaction; bacteria or firefly luciferases, which catalyze a photoreaction Enzymes; enzymes such as glycosyltransferases that form non-protein structures that are readily detected by antibodies, lectins or cognate binding proteins; corresponding antibodies or recognition proteins are well known Proteins that are easily monitored during cell surface expression or secretion, such as surface or secreted antigens; catalyze the synthesis of fluorescent structures in the absence of exogenous substrates, or stoichiometrically embody fluorescent structures And proteins such as jellyfish fluorescent protein (eg, GFP).

“Operably linked” means that the reporter protein gene is located adjacent to a promoter that transcribes the gene and ultimately drives expression of the reporter protein.

“Promoter” means any minimal nucleic acid sequence sufficient to drive transcription of a reporter gene. A promoter is one that is activated by binding to a polypeptide. An example of a promoter useful in the present invention is a promoter that normally binds to a gene that is expressed when the cell is in an abnormal or diseased state (eg, due to cancer, inflammation or bacterial or viral infection), and whose protein product is in this state. Is a direct or indirect cause of Suitable promoters include, but are not limited to, the NF-kB promoter, the interleukin-2-promoter and the HIV-1 long terminal repeat promoter.

[0030] A DNA molecule library refers to a set of DNA molecules, each within a DNA expression vector. Preferably, the DNA expression vector exhibits high efficiency such that the expression level is high. In most cases, the library will contain DNA molecules that encode dozens, hundreds or thousands of different polypeptides, but the DNA molecules in the library may encode only one polypeptide ( For example, in the last stage of sib selection). A library of hundreds of different DNA molecules is considered to be more "complex" than a library of five different DNA molecules. Like DNA libraries, compound libraries can have varying degrees of complexity. One feature of the invention is a method that allows tracking of the activity of interest by sequential screening of a less complex library.

“Candidate compound” means a natural or artificial compound that is being investigated for its ability to modulate BCMA or NF-kB biological activity. Candidate compounds may include, for example, peptides, polypeptides, antibodies (and fragments thereof), synthetic organic molecules, natural organic molecules, nucleic acid molecules, and components or derivatives thereof. Candidate compounds include animals,
Cells, lysates or extracts derived from cells, or molecules derived from cells can be used to screen using any of the methods described herein. Measurements include, for example, altered gene expression, altered
RNA stability, altered protein stability, altered protein levels,
It may be for the purpose of detecting altered protein phosphorylation or biological activity of the altered protein. Means for measuring may include, for example, antibody labeling, immunoprecipitation, phosphorylation assays and methods well known to those skilled in the art for detecting nucleic acids.

“Modulate” means a change either due to a decrease or an increase.

By “BCMA polypeptide” is meant a polypeptide that exhibits substantial identity to a human or mouse BCMA shown in FIGS. 7A and 7B, respectively, over a region of 20 contiguous amino acids and has BCMA biological activity. I do.

“Substantially identical” refers to a sequence identity of at least 80% when two polypeptide sequences are optimally aligned, such as by the GAP or BESTFIT programs using default gap weights. And more preferably at least 90
% Sequence identity, most preferably at least 95% or more than 99% sequence identity.

By “heterologous compound” is meant, in the context of the present invention, a polypeptide, compound or detectable label (eg biotin) that does not naturally bind to the polypeptide.

“BCMA biological activity” means BCMA-mediated modulation of NF-kB biological activity or expression or binding of an antibody that specifically binds to a BCMA polypeptide.

“NF-kB biological activity” refers to any function performed by activated NF-kB.

The invention features a method of screening DNA and compound libraries for the ability to modulate reporter gene expression in cells expressing a recombinant anti-cell death gene. If the gene is not expressed by the expression of this gene,
Cells can survive under conditions that result in cell death or consequent failure of the screening procedure.

The method of the present invention is suitable for identifying a polypeptide that regulates transcription from a selected promoter. Also, these polypeptides target drugs. The methods of the present invention are also suitable for identifying compounds that themselves mimic or block the function of a polypeptide that regulates the transcription of a selected promoter.

Accordingly, the invention features a novel and efficient multi-step method for identifying lead compounds that regulate expression from a promoter to develop pharmaceutical compounds.

[0041] The invention also features a novel NF-kB activator, BCMA. Since NF-kB is involved in many cellular processes and disease states, BCMA is useful for treating disease. BCMA is also useful for identifying compounds that modulate its expression or biological activity. These compounds can be developed as drugs and used as lead compounds for the purpose of drug identification.

[0042] Other features and advantages of the invention will be apparent from the following detailed description, drawings and claims.

DETAILED DESCRIPTION The present invention provides methods for the rapid identification of molecules involved in the regulation of specific signaling pathways. It can be used both as a target identification tool and as a rapid assay for drug effects, and by itself can significantly reduce the amount of time required to go from target to hit.

The method features an expression cloning method that identifies a polypeptide capable of activating a reporter gene (FIG. 1). Such a polypeptide is referred to herein as an activating polypeptide (FIG. 1). Expression cloning is a technique for identifying polypeptides based solely on their ability to produce an observable activity of interest.
For this activity to be identified, the polypeptide must be widely expressed in vivo or in vitro, and after the polypeptide has been expressed, a reasonably sensitive assay must be available to detect the activity. . In this method, it is desirable that the activity detected depends on the output of the reporter gene (FIG. 1).

Although the use of expression cloning in conjunction with a reporter gene is not new, current applications have not resulted in a highly efficient system for the identification of many new molecules. This method is a high productivity system for the identification of cDNA clones encoding polypeptides that induce reporter gene activity. Some features of the high productivity system of the present invention include (1) a very efficient cDNA expression vector;
A) a method for introducing large amounts of plasmid into a plurality of cells; (3) a feature for preventing reporter cell death due to toxicity of the expressed gene;

Expression cloning can be performed by either of two common instances, positive selection and sibling selection, also known as pool division. Positive selection is the process of physically separating a cell or a collection of genetically linked proteins from a population that encodes a much larger number of cells or molecules of little interest, resulting in a cell, virus, or genetic And methods for enrichment of nucleic acids encoding them. Since the nucleic acid encoding the polypeptide of interest is physically associated with the cell, virus or other population they contain, it is physically purified by positive selection, a step in which the activity of interest is reduced. This can be repeated until a single encoding nucleic acid is found. In a positive selection strategy, it is desirable to develop a method for the association of a single nucleic acid with a single protein assembly or cell. Thus, in positive selection, it is desirable to identify and use a method for introducing nucleic acids into cells that results in a single nucleic acid species per cell.

The method of the present invention uses sibling selection, a screening method that assayes a pool of molecules from a cognate library of nucleic acids for activity and detects a positive library by some signal (eg, reporter gene expression). . afterwards,
The nucleic acids that make up the library are separated into less complex libraries, which are then re-assayed and subdivided until a single nucleic acid encoding a polypeptide with the desired activity is found (FIGS. 2, 3, and Four). Since sibling selection relies on detecting the activity of a collection of nucleic acids, the result is a large number of nucleic acid species per cell,
It is often advantageous to use methods for transfection or introduction into cells. The sibling selection scheme can also be performed entirely in vitro, as in the positive selection method.

Features of the Invention Transfection Method In order for the present invention to make sibling selection an efficient method for cloning genes, it is important that a large number of DNA molecules be introduced into each cell. Many methods for introducing DNA molecules are known in the art, including microinjection, complexation with positively charged synthetic polycations such as DEAE dextran, polybrene, polylysine or polyarginine, histone or other basic Complexation with proteins, complexation with cationic lipids or related amphipathic molecules, condensation with polyethylene glycol or polyhydroxybutyrate, coprecipitation with calcium phosphate, electroporation, scrape loading (scrape) loading) or partial rupture, and fusion with bacterial or microbial spheroplasts. Of these, those that can be easily performed in parallel and that result in the co-introduction of multiple nucleic acids encoding different species into reporter cells are preferred. Further, the method of transfection will preferably provide on average at least 25 DNA molecules per cell. More preferably, the average number will be at least 100 or just 500 DNA molecules per cell. Methods to achieve favorable results include calcium phosphate coprecipitation, complexation with polycations or cationic lipids, and condensation with uncharged polymers such as polyethylene glycol.

A library of nucleic acid molecules encoding potentially activating polypeptides can also be created in a biologically active population, such as a virus or a virus transmitting particle capable of introducing itself directly into a reporter cell. . In such cases, cD
The NA library is prepared in a viral vector, and the library of active viruses or transmitting particles is applied to reporter cells. Generally, the reporter cell will have been previously engineered to contain the reporter gene, but the reporter gene may be introduced simultaneously with the activating nucleic acid.

Highly Efficient cDNA Expression Vector In order to detect ectopic activation of a signal transduction pathway, it is desirable to greatly express a protein acting as a signal transduction intermediate.

The present invention uses a highly efficient cDNA expression system to produce proteins in reporter cells. Such systems include, for example, elongation factor 1 alpha (EF-1α
), The 3 'untranslated region (3'UTR) and polyadenylation consensus (poly (A)) sequences from the human growth hormone gene, and the use of a very strong promoter such as the human IgGl hinge-CH2 intron. Other strong promoter and non-promoter elements are known in the art (eg, mouse or human cytomegalovirus immediate early gene promoter, globin intron and 3 ′ UTR / poly (A) sequence).

Cell Death Inhibition Another important feature of the present system is a method for preventing the death of cells that overexpress the protein. Such an approach is important for two reasons. High levels of signaling intermediates themselves can lead to cell death,
Also, when a library of nucleic acids is transfected, the presence of a nucleic acid encoding a toxic protein, even at low frequencies, can prevent detection of a desired signal. The latter effect can be expected to have a greater impact as the size of the library increases.

Several methods are known for preventing programmed cell death or death of cells undergoing apoptosis. Both viral and cellular antagonists of cell death are known, some species acting upstream of the apoptotic pathway or at multiple stages. Examples of the former include dominant negative forms of cell death proteins, such as FADD or TRADD, and cellular inhibitors of apoptosis, such as members of the viral or cellular IAP family. In several stages,
Examples of proteins that act or are relatively downstream are the orthopoxvirus CrmA and baculovirus p35 proteins, members of the cellular Bcl family, and cysteine proteases that act in the zymogen cascade to generate a program of death. Includes peptidic and non-peptidic inhibitors of caspases. To prevent apoptosis in expression cloning, CrmA and
Combinations of a wide range of anti-apoptotic proteins, such as Bcl-xL, with different mechanisms of action are preferred.

[0054] Identification of the inventors of the gene product results in the activation of Example transcription factor NF-kB, the reporter cells by stably transfected the following two constructs commercial human embryonic kidney cell line 293 EBNA The strain was established: a reporter construct consisting of the NF-kB promoter element upstream of green fluorescent protein (GFP); and a eukaryotic expression construct expressing two anti-cell death molecules, CrmA and Bcl-xL. The former serves as an indication of the presence of a signal that activates the NF-kB signaling pathway, while the latter prevents these cells from undergoing programmed cell death in the presence of a pro-apoptotic signal I do. Receptor (
For example, when NF-kB activating molecules, including tumor necrosis factor α receptor 1), ligands (eg, tumor necrosis factor α), and intracellular signaling proteins (eg, RIP) are expressed in a reporter cell line, GFP A strong production of occurs. GFP production levels as a measure of NF-kB activation can be assayed using fluorescence microscopy or flow cytometry. High level expression of these various NF-kB activating molecules
The DNA was obtained by placing the DNA under the control of the very strong EF-1α promoter present on the commercially available plasmid PEAK8 (Edge Biosystems). Thus, these cells can be transfected with a cDNA library to identify individual cDNAs that can activate NF-kB.

The cDNA library prepared from activated human T cell mRNA and cloned into the PEAK8 vector was split into smaller libraries of approximately 500 cDNA clones each and DNA was prepared from each library. DNA from the library is analyzed by calcium phosphate precipitation according to published protocols, eg, Ausubel et al. (1997) “Current Protocols in Molecular Biology”, Willie Interscience, by reporter cell line. Was transfected. Cells were grown for 48 hours and assayed for GFP production. CDNA libraries that produced a positive GFP signal, defined by the appearance of at least 0.1% of bright cells by fluorescence microscopy, were further screened by subdividing each cDNA into 50 libraries (FIG. 2). DNA was prepared from each library, transfected into a reporter cell line, and assayed for GFP production. The process was repeated until one cDNA clone displaying positive GFP was obtained. By screening techniques, known NF-kB activating molecules such as surface receptors (DR3, FAS), soluble ligands (interleukin-1, TRAIL, CD40 ligand), and intracellular signaling molecules (low molecular weight GTPase rho) DNA to encode
Was cloned. Previously known molecules and new molecules without known function (BCMA) were also identified.

The inclusion of two anti-cell death genes in the transfected cells is described in D above.
It may be useful in expression cloning of at least some of the NAs. It has been found that expression of either FAS or DR3 in a cell induces apoptosis in that cell. The fact that each is recovered demonstrates the improved properties of the expression cloning method of the present invention. Similarly, the method of the present invention preferably employs a high copy number so that otherwise potentially dead cells can survive by expression of the anti-cell death gene by multiple inserts. .

The method is applied more quickly and effectively using labeled cDNA bacterial culture trains, where each culture is derived from a single colony and thus independently derived cDNA Represents the expression plasmid (FIG. 5). Assess the relative activity of every row and every column by pooling small cultures from large array rows and columns and preparing DNA from each of the libraries, thereby dramatically reducing effort Is possible (Fig. 6). For example, if 90,000 clones / clone are arranged in a 300 × 300 matrix, transfecting 300 rows and 300 columns will determine all rows and all columns with positive clones. Thus, the array displays only 600 DNA preparations and transfections. If there is only one positive culture, the operation is completed. If there is a large number of positive cultures, at worst, each row and each column will have only one positive cell.
The positive cells if x number exists, possibly some intersection exists ^two X. As long as x is a small number, the work required is quite modest. For example, if x is 10, clone 9
A total array of 0,000 can be screened in as few as 700 DNA preparations and transfections.

Variations of the experimental approach outlined above are applicable depending on the particular system to be examined. Reporter systems and cell lines can be adapted for the promoter to be examined. For example, replacing the NF-kB promoter in the GFP reporter construct with an interleukin-2 promoter and introducing it into a T lymphocyte cell line to screen for molecules that regulate signaling pathways leading to interleukin-2 expression in T lymphocytes can do. Other promoters are HIV-1 long terminal repeats (L
TR) promoter, or other inducible promoters. The calcium phosphate transfection technique can be modified to another method that is more suitable for a particular reporter cell type. Another transfection protocol can be based on electroporation, cationic lipids, DEAE dextran, spheroblast fusion or virus-mediated delivery.

A variation of the above method is to screen for molecules that terminate expression from the promoter. For example, NF-kB receptors in reporter cell lines are activated in response to interleukin-1 stimulation. Interleukin-1 in culture medium
By adding, a molecule that inhibits interleukin-1-dependent expression from the NF-kB promoter can be searched. Depending on the cDNA library used,
These molecules can be the original negative regulatory molecules or mutants that exhibit dominant negative behavior. Similarly, screening can be performed on combinatorial libraries to look for small pharmacological molecules that negatively interfere with the pathway (see below).

The above screening methods are well suited for screening genes from a particular organism that interact with pathways in another organism. A prime example is the screening of expressed genomes of viruses, bacteria, or other pathogens for genes that, when expressed, may interact with the NF-kB pathway. This method of using these pathogens to alter the immune response to strengths can identify potential targets for pharmacological intervention.

[0061] The reporter cell may also provide a protein that increases the effectiveness of the reporter gene. These proteins are usually introduced by transfection of the expression plasmids that encode them. A protein that increases the effectiveness of a reporter gene may provide an essential element of a signaling pathway that would not otherwise be present in a reporter cell, or incorporate, amplify, or select for a signal from the pathway. It may be composed of an artificial protein that acts to react specifically. Of particular relevance are artificial transcriptional activities, which consist of a DNA-binding element that specifically interacts with the recognition binding site in the artificial promoter and a pathway-specific transcriptional activator that responds to activation of pathways that have altered transcriptional activity. Is a conversion factor. Such artificial activators are known in the art as a fusion of a bacterial or yeast DNA binding protein with a mammalian transcription factor activation domain. Such an activation domain provides a protein binding site, an altered ligand-activated configuration, or a post-translational modification site that increases the ability of the artificial activator to promote transcription. Examples of commercially available or known artificial activators in the art include LexA and c-Jun, Elk1, CREB, c-Fos, ATF2, CH2
OP4 and Gal4 fusions with members of the nuclear hormone superfamily are included.

In one example, (i) a recombinant anti-cell death gene, (ii) a chimeric transcription factor consisting of a Gal4 DNA binding domain fused to a c-Jun activation domain, and (iii) a basal promoter and Gal4 binding Cells containing the GFP-encoding DNA operably linked to the regulatory sequences comprising the sites are used to identify polypeptides or compounds that regulate c-Jun activation. For example, if c-Jun phosphorylation (ie, activation) occurs due to polypeptide expression, an increase in GFP expression would occur.

BCMA is an activator of NF-kB Using the methods described herein, we have identified BCMA as an activator of NF-kB. BCMA was discovered by molecular analysis of the t (4; 16) translocation feature of human T-cell lymphoma (Laabi et al., EMBO J. 11: 3897-3904, 1992), and its function has been previously unknown. Was.

Based on this discovery, BCMA is a target for drug discovery or rational drug design. For example, compounds that modulate BCMA expression or biological activity will also modulate NF-kB biological activity. Accordingly, the invention features methods and reagents for identifying NF-kB modulating compounds.

[0065] BCMA polypeptides or nucleic acid molecules are also useful for treating diseases associated with insufficient or inappropriate NF-kB biological activity or expression. A BCMA polypeptide or nucleic acid molecule is administered to a patient using an appropriate transporter known in the art. Generally, a BCMA polypeptide or nucleic acid molecule will be delivered in a pharmaceutically acceptable carrier.

Identification of Coupled Targets and Generation of Assays The identification of molecules that may play a role in modulating inflammation and tumorigenesis is based on NF-kB
Directly related to signaling pathways. Research on NF-kB activating molecule
It should not be limited to screening expression libraries. Compounds that modulate the products of the pathway can be identified by mimicking the activity of the polypeptide using the same principles, or inhibiting the activity of the polypeptide if it is overexpressed. The latter compound is believed to act on or downstream of the overexpressed protein. Once sufficient activating proteins have been identified, it is possible to identify at which stage the compound acts by assessing their effects on a panel of transfected cells expressing different activating proteins. It is considered to be.

In general, compounds can be obtained from natural and synthetic sources according to methods known in the art.
Or semi-synthetic) extracts from both large libraries or chemical libraries. One skilled in the art of drug discovery and development will understand that the exact origin of the test extract or compound is not critical to the screening technique (s) of the present invention. Thus, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant, fungal, prokaryotic, or animal based extracts, fermentation broths, and modifications of existing compounds along with synthetic compounds. Many compounds are also useful for performing random or directed synthesis (eg, semi-synthetic or total synthesis) of any number of chemical compounds, including but not limited to compounds based on carbohydrates, lipids, peptides and nucleic acids. Available. Synthetic compound libraries are available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available from Biotics (Sussex, UK), Xenova (Slow, UK), Harbor Branch Ocean Graphics It is available from a number of sources, including the Harbor Branch Oceangraphics Institute (Ft Pierce, FL), and PharmaMar America (Cambridge, Mass.). In addition, natural and synthetically generated libraries may be produced, if desired, according to methods known in the art, for example, by standard extraction and fractionation methods. Further, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

Further, those skilled in the art of drug discovery and development will recognize that replication (eg, taxonomic replication,
Biological elimination, and chemical elimination, or any combination thereof), or elimination of duplicates or repetitive units of material already known to regulate the test promoter, if possible. Easy to understand.

If the crude extract is found to regulate reporter gene expression, a further fractionation of the positive lead extract is required to isolate the chemical constituent responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of chemical entities within the crude extract having the desired activity. Methods for fractionating and purifying such heterologous extracts are known in the art. If desired, compounds that have been shown to be useful agents for modulating gene expression or biological activity are chemically modified according to methods known in the art.

Primary Screening of Compounds that Modulate BCMA Biological Activity Modulating NF-kB expression or biological activity regulates many important cellular processes. The finding that NF-kB activity is regulated by BCMA allows us to provide assays for agents that modulate NF-kB by monitoring BCMA expression or biological activity. In such an assay, BCMA expression may be measured by measuring changes in:
(b) level of BCMA RNA; (c) level of BCMA-mediated NF-kB biological activity; or (d) level of a reporter gene or protein expressed from the NF-kB promoter. These measurements may be made in vitro or in vivo. These assays can identify compounds that modulate NF-kB biological activity (eg, gene transcription). Compounds so identified are, for example,
It may have therapeutic value in treating diseases with too little or too much cell death.

[0071] Cells that overexpress BCMA can be produced using standard techniques. The identified compound may bind to BCMA and block NF-kB BCMA activation. Compound screening can be performed on primary cultured cells, but cell lines can be used as well. Cell lines can be modified so that the cells constitutively express a BCMA polypeptide, eg, a BCMA intracellular domain.

[0072] Any cell line, such as the cells described above, can be engineered (as described above) to include a reporter gene expressed under the control of the NF-kB promoter. A preferred reporter gene encodes GFP. Typically, gene expression (eg,
Endogenous NF-kB gene, or a recombinant reporter gene expressed under the control of the NF-kB promoter or a fragment thereof) is measured by assaying the RNA or protein levels of the expressed gene or both. . For example, NF-kB
The polypeptide expressed by the gene or reporter gene produces a detectable signal under conditions where compound-mediated changes are measured. To quantitatively determine the amount of signal, as described herein, the amount of signal generated in the absence of any compound to be tested is generated when the cell is contacted with the compound. Need to be compared to the amount The comparison allows the compound to be identified as a compound that causes a change in the detectable signal (eg, at the RNA or protein level) produced by the expressed gene, and thus modulates NF-kB expression Identify compounds that can. To prevent NF-kB cells from dying, a second gene encoding an apoptosis inhibitor can also be expressed in the cells, as described herein.

Secondary Screening for Compounds that Modulate NF-kB Activity Once test compounds that appear to modulate NF-kB expression have been identified, it may be necessary or desirable to conduct further tests on these compounds. Can be The present invention provides such a secondary confirmation assay. For example, a compound that appears to modulate NF-kB activity in an earlier test may be subjected to an additional assay to confirm that the compound also modulates NF-kB activity in vivo. In the first round of in vivo testing, NF-kB activity is initiated in the animal by well-known methods and the compound is administered by one of the means described in the "Treatment" section below. Cells or tissue are isolated within hours to days after injury and subjected to assays to assess NF-kB expression or biological activity levels. Such assays are well-known to those skilled in the art. Examples of such assays include ELISA, Western blot analysis, RT-PCR
, RIA, and Northern blot analysis.

Therapy NF-kB is a key regulator of inflammatory responses (eg, rheumatoid arthritis, inflammatory bowel disease, septic shock), tumorigenesis, and antiviral and antimicrobial responses. Thus, the discovery of new gene products that regulate NF-kB activity and thus regulate disease processes will identify molecular targets for pharmacological intervention. By increasing or mimicking BCMA biological activity
For example, it can enhance anti-tumor antibody production or increase T cell toxicity to tumor cells in cancer immunotherapy. Conversely, antagonizing BCMA biological activity may be advantageous, for example, in situations where it is desirable to down regulate immune cell function. A compound identified by any of the methods disclosed herein may be administered to a patient or experimental animal in a unit dosage form together with a pharmaceutically acceptable diluent, carrier, or excipient. For administering such compositions to a patient or experimental animal, conventional pharmaceutical practice may be used to provide a suitable formulation or composition. Intravenous administration is preferred, for example, parenteral, subcutaneous, intramuscular,
Any suitable route of administration may be used, such as intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic dosage forms may be in the form of liquids or suspensions, for oral formulations, the formulations may be in the form of tablets or capsules, and for intranasal formulations, powders,
It may be in the form of nasal drops or aerosol.

Methods well known in the art for making formulations are described, for example, in Remington
ngton) "The Science and Practice of Pharmacy" No. 1
Recognized in the 9th Edition, Gennaro, AR, eds. 1995, Mac Publishing Company, Easton, PA. Formulations for parenteral administration include, for example,
Excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated naphthalene may be included. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compound. Other potentially useful parenteral delivery systems for the antagonists or agonists of the invention include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The inhalation formulation may contain excipients, for example lactose, or may be an aqueous solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or in the form of nasal drops, Alternatively, it may be an oily solution for administration as a gel.

As described herein, the present inventors have discovered that BCMA activates NF-kB activity. NF-kB then activates a number of cellular processes. Therefore, NF-
Any compound that modulates kB expression is a candidate compound for use in drug development. One potential compound is a polypeptide fragment of BCMA that retains the ability to bind another component of the signaling pathway, but has lost the ability to activate NF-kB. Such polypeptides are believed to act as inhibitors of wild-type NF-kB signaling. Another potential compound is a polypeptide fragment of BCMA that exhibits constitutive activation of NF-kB.

Other Embodiments All publications and patent applications mentioned in this specification are to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. , Incorporated herein by reference.

Although described in relation to particular embodiments thereof, further modifications may be made to the invention, and the present application is intended to cover, in general, any alterations, uses, or adaptations of the invention, including the principles of the invention. It is understood that such deviations are within the known or conventional practice within the art to which this invention belongs and are construed to apply to the essential features described herein above. It should be.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a reporter cell system for rapid identification of cDNA encoding a polypeptide of interest. A promoter (in this case, NF-kB
A reporter gene (eg, GFP or T) operably linked to a promoter.
Cells containing hy-1) were transfected with the cDNA library. If the polypeptide expressed by one of the plasmids in the reporter cell is capable of inducing expression from the NF-kB promoter, the reporter gene is expressed and a detectable marker protein is produced.

FIG. 2 is a schematic diagram of a cDNA library divided into smaller libraries. Thereafter, each library is transfected into reporter cells. Thereafter, the library that induces reporter gene expression is further divided until a cDNA encoding a polypeptide that induces reporter gene expression is isolated.

FIG. 3 is a schematic diagram of three stages of enrichment for an intracellular signaling intermediate. The ratio indicates the appearance rate of the cDNA encoding the polypeptide that induces the reporter gene, compared to the total cDNA. Note that during transfection, each cell will receive multiple plasmids.

FIG. 4 is a schematic showing how secreted factors can be distinguished from intracellular signaling intermediates such as receptors or kinases. The soluble ligand diffuses through the medium and binds to the receptor on untransfected cells. This leads to expression of the reporter gene in most, if not all, cells. Intracellular effectors are restricted to transfected cells.

FIG. 5 is a schematic diagram of an arrayed clone. N ² elements pooled in N rows and N columns can be assayed in 2N transfections. In the example provided, 4
Rows and 4 columns were positive. In this case, in order to identify unambiguously intersections are required 4 ^twice i.e. 16 transfections.

FIG. 6 is a schematic diagram of an indexed library protocol. The method is similar to the method described in FIGS. 1-5, except that the plasmid is grown as an individual culture. Thereafter, a library of plasmids is prepared and assayed. This method facilitates rapid recovery of individual plasmids and prevents loss of positives when the library is split into low complexity libraries.

FIG. 7A: Sequence of human BCNA polypeptide (GenBank accession number Q02)
FIG. 223) is a schematic view showing (223). Putative transmembrane domains are shown in bold.

FIG. 7B: Sequence of mouse BCNA polypeptide (GenBank accession number A
AC23799).

FIG. 8 is a schematic diagram showing that the intracellular domain of human BCMA can activate NF-kB. A series of fusion proteins were made using the CD5L leader sequence, IgG Fc, CD7 transmembrane domain and amino acids from human BCMA (SEQ ID NO: 1) as follows: Ig7bcma: amino acids 78-184; ΔC20: amino acids 78 -164; ΔC40: amino acids 78-144; ΔC60: amino acids 78-124; ΔC80: amino acids 78-104; ΔN20: amino acids 9
8-184; ΔN40: amino acids 118-184; ΔN60: amino acids 138-184; ΔN80: amino acids 15
8-184.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/47 C12Q 1/02 4H045 14/705 1/68 Z C12N 5/10 G01N 33/15 Z C12Q 1 / 02 33/50 Z 1/68 33/566 G01N 33/15 33/68 33/50 C12N 15/00 ZNAA 33/566 5/00 B 33/68 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ) , BY, KG, KZ MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF term (reference) 2G045 AA40 DA36 4B024 AA01 BA63 CA01 FA02 GA11 HA20 4B063 QA18 QQ79 QR32 QR33 QR33 QR72 QS24 4B065 AA90X AB01 AC14 BA01 BA24 BB46 4C084 AA02 BA44 NA14 ZB112 ZB262 ZB332 4H045 AA10 BA10 CA11 DA50 FA74

Claims (44)

[Claims] 1. A method for identifying a polypeptide that increases gene expression from a promoter, comprising: (a) a library comprising a polypeptide that increases gene expression from the promoter. Contacting a polypeptide library with cells containing a reporter gene operably linked to the promoter, wherein the recombinant anti-cell death gene is expressed such that expression of the reporter gene is increased, if any; A) determining whether expression of said reporter gene in said cells is increased as a result of contacting said polypeptide library; and (c) determining if said reporter gene expression is increased, expression of said reporter gene. Identifying increased polypeptides of the library. 2. The method of claim 1, wherein step (c) comprises the following steps: (i)
Dividing the library into two or more less complex libraries; and (ii) repeating steps (a) and (b) until a polypeptide that activates reporter gene expression is identified. . 3. A method for identifying a polypeptide that reduces gene expression from a promoter, comprising: (a) the library comprises a polypeptide that reduces gene expression from the promoter. Contacting a polypeptide library with cells containing a reporter gene operably linked to said promoter, wherein said polypeptide expresses a recombinant anti-cell death gene such that expression of said reporter gene is reduced, if any; A) determining whether expression of said reporter gene in said cells is reduced as a result of contacting said polypeptide library; and (c) reducing expression of said reporter gene if reporter gene expression is reduced. Identifying the polypeptide to be effected. 4. The method of claim 3, wherein step (c) comprises the following steps: (i)
Dividing the library into two or more less complex libraries; and (ii) repeating steps (a) and (b) until a polypeptide that reduces reporter gene expression is identified. 5. A method for identifying a polypeptide that alters activation of a transcription factor activation domain, comprising the steps of: (a) expressing a reporter gene, wherein gene expression from a promoter is A yeast or bacterium fused to a recombinant anti-cell death gene and a mammalian transcription factor activation domain, wherein the polypeptide is altered when the library alters the activation of the transcription factor activation domain is included in the library. A library of polypeptides, which expresses a chimeric transcription factor comprising a sex DNA binding domain, and also comprises a basal promoter and a reporter gene operably linked to the promoter comprising the binding site of the DNA binding domain; and a library of polypeptides. (B) as a result of contacting with the polypeptide library, the reporter gene in the cell Step expression to determine whether they have changed; when the expression of and (c) a reporter gene is changed, the step of identifying a polypeptide that alters the reporter gene expression. 6. The method of claim 5, wherein step (c) comprises: (i)
Dividing the library into two or more less complex libraries; and (ii) repeating steps (a) and (b) until a polypeptide that alters reporter gene expression is identified. 7. The method of claim 1, 3, or 3 wherein the contacting comprises expressing a library of DNA molecules in a cell such that the library of DNA molecules encodes a polypeptide library. 6. The method according to claim 5. 8. The cell according to claim 7, wherein the cell expresses a recombinant anti-cell death gene and contains a reporter gene operably linked to a promoter.
The described method. 9. The method of claim 7, wherein the library of DNA molecules is introduced into the cells by transfection. 10. The method of claim 9, wherein the average number of DNA molecules introduced into the cells by transfection is at least 25. 11. The method of claim 9, wherein the average number of DNA molecules introduced into the cells by transfection is at least 100. 12. The method of claim 9, wherein the average number of DNA molecules introduced into the cells by transfection is at least 500. 13. The method of claim 7, wherein the DNA molecule is expressed from a high efficiency expression system. 14. The polypeptide of claim 1, 3 or 3 wherein said polypeptide expresses a recombinant anti-cell death gene and is similar to a cell containing a reporter gene operably linked to a promoter. 6. The method according to claim 5. 15. The polypeptide of claim 1, 3 or 3 wherein the polypeptide expresses a recombinant anti-cell death gene and is different from a cell containing a reporter gene operably linked to a promoter. 6. The method according to claim 5. 16. The method according to claim 1, wherein the polypeptide is selected from the group consisting of an extracellular ligand, a cell surface receptor, and a signaling intermediate. . 17. A method for identifying a compound that alters gene expression from a promoter, comprising the steps of: (a) If the library contains a compound that alters gene expression from a promoter. Contacting a compound library with cells containing a reporter gene operably linked to the promoter, wherein the compound library expresses a recombinant anti-cell death gene such that the expression of the reporter gene is altered; Determining whether expression of the reporter gene in the cell has changed as a result of contacting the rally; and (c) increasing expression of the reporter gene if the reporter gene expression has changed. Identifying the compounds in the library. 18. The method of claim 17, wherein step (c) comprises: (i)
) Dividing the library into two or more less complex libraries; and (ii) repeating steps (a) and (b) until a compound that alters reporter gene expression from the promoter is identified Stage. 19. The method according to claim 1, wherein the promoter is a heterologous promoter.
18. The method according to any one of claims 3, 5, 5 or 17. 20. The method according to claim 1, wherein the promoter is derived from a mammal, and the polypeptide library comprises a polypeptide derived from a bacterium or a virus.
18. The method according to any one of claims 5 or 17. 21. The method according to claim 1, wherein the reporter gene is GFP. 22. The method according to claim 1, wherein the anti-cell death gene is selected from the group consisting of a member of the bcl family, a member of the IAP family, and crmA. The method described in the section. 23. The cell according to claim 1, wherein the cell is selected from the group consisting of CHO cells, CD-1 cells, Cos cells, 293 cells, HeLa cells, BHK cells, and L cells. Or a method according to any one of claims 17 to 17. 24. The candidate compound is expressed as NF-kB by altering the expression level of the BCMA polypeptide in the cell as compared to a cell that has not been contacted with the candidate compound.
A method for determining whether a compound alters NF-kB biological activity, such as identifying a compound that alters biological activity, comprising the steps of: a) expressing a BCMA polypeptide Supplying the cells; b) contacting the cells with a candidate compound; and c) measuring the expression level of the BCMA polypeptide in the cells. 25. The method according to claim 23, wherein the BC is compared to a polypeptide that has not been in contact with the candidate compound.
A method of determining whether a compound alters BCMA biological activity, such as identifying the candidate compound as a compound that alters BCMA biological activity by altering the level of biological activity of the MA polypeptide, comprising: A method comprising the steps of: a) providing a BCMA polypeptide; b) contacting the cells with a candidate compound; and c) measuring the level of biological activity of the BCMA polypeptide. 26. The method of claim 25, wherein the BCMA polypeptide is present in a cell. 27. The method of claim 25, wherein the BCMA polypeptide is in a cell-free system. 28. The method of claim 25, wherein the BCMA biological activity is a modulation of NF-kB biological activity. 29. The method of claim 28, wherein the NF-kB biological activity is a modulation of cell death. 30. The BCMA polypeptide is a human BCMA (SEQ ID NO: 1) at positions 98-16.
26. The method of claim 25 comprising an amino acid at position 4 or a polypeptide sequence having substantial identity to amino acids 97-163 of mouse BCMA (SEQ ID NO: 2). 31. A method for determining whether a compound alters NF-kB biological activity, such that the candidate compound that binds to the polypeptide is a compound that alters NF-kB biological activity, comprising: A method comprising: a) providing a BCMA polypeptide; b) contacting the polypeptide with a candidate compound; and c) detecting binding of the candidate compound to the polypeptide. 32. The polypeptide is substantially identical to amino acids 98-164 of human BCMA (SEQ ID NO: 1) or amino acids 97-163 of mouse BCMA (SEQ ID NO: 2). 32. The method of claim 31, comprising a polypeptide sequence having 33. It has substantial identity to amino acids 98 to 164 of human BCMA (SEQ ID NO: 1), and has amino acids 1 to 54 of human BCMA (SEQ ID NO: 1). A substantially pure polypeptide comprising no polypeptide sequence. 34. The polypeptide according to claim 33, which alters NF-kB activity. 35. The polypeptide according to claim 33, comprising the amino acids 98 to 164 of human BCMA (SEQ ID NO: 1). 36. It has substantial identity to amino acids 97-163 of mouse BCMA (SEQ ID NO: 2) and has amino acids 1-49 of mouse BCMA (SEQ ID NO: 2). A substantially pure polypeptide comprising no polypeptide sequence. 37. The polypeptide of claim 36, which alters NF-kB activity. 38. The polypeptide according to claim 36, comprising the amino acids 97-163 of mouse BCMA (SEQ ID NO: 2). 39. The method of claim 33 or 36, wherein the compound is covalently linked to a heterologous compound.
An NF-kB modulator comprising the described polypeptide. 40. The modulator according to claim 39, which alters NF-kB activity. 41. A method for activating NF-kB activity in a cell, the method comprising contacting the cell with a recombinant BCMA polypeptide having NF-kB activating activity. 42. A method of activating NF-kB activity in a cell, comprising contacting the cell with a recombinant nucleic acid molecule encoding a recombinant BCMA polypeptide having NF-kB activating activity. Method. 43. Use of a BCMA polypeptide for preparing a pharmaceutical composition for treating cancer, apoptosis, viral infection, or an inflammatory response. 44. Use of a BCMA nucleic acid molecule for preparing a pharmaceutical composition for treating cancer, apoptosis, viral infection, or an inflammatory response.